Staphylococcus aureus (SA) is a Gram-positive human pathogen that causes a wide range of infections from skin and soft tissue infections (SSTI) to life threatening sepsis and pneumonia. It is a leading cause of hospital- and community-associated infections worldwide (Barrio, et al., 2006, Microbes Infect, 8 (8):2068-2074; Brown, et al., 2009 Clin Microbiol Infect, 15 (2): 156-164; Colin, et al. 1994, Infect Immun, 62 (8):3184-3188). The range of pathologies reflects the diverse abilities of SA to escape the immune response using a plethora of virulence factors: the superantigenic and pore-forming toxins, coagulase, capsular polysaccharide, adhesins, proteases, complement inactivating exoproteins, and other innate response modifiers (Barrio, et al., 2006, Microbes Infect, 8 (8):2068-2074; Deurenberg and Stobberingh, 2008, Infect Genet Evol, 8 (6):747-763).
Since its first emergence in the 1960s methicillin-resistant SA (MRSA) has become endemic in healthcare settings worldwide (Diep, et al., 2006, J Infect Dis, 193 (11):1495-1503). Since the 1990s, community associated MRSA strains (CA-MRSA) emerged, and are posing a major global challenge (Bassetti, et al., 2009, Int J Antimicrob Agents, 34 Suppl 1:S15-19; Bradley, 2005, Semin Respir Crit Care Med, 26 (6):643-649; Chambers, 2005, N. Engl J Med, 352 (14):1485-1487.). Alpha hemolysin (α-toxin, Hla) is a major virulence factor in SA pneumonia and SSTI (Bubeck Wardenburg and Schneewind, 2008, J Exp Med, 205 (2):287-294; Kennedy, et al., 2010, J Infect Dis, 202 (7):1050-1058). Recently, cytolytic short peptides known as phenol soluble modulins (PSMs) were identified as key virulence factors that lyse neutrophils, the main line of defense against S. aureus (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). Another related cytolytic short peptide of staphylococci is known as delta hemolysin or delta toxin (δtoxin) the key marker of S. aureus quorum sensing system (agr) (Novick, et al., 1993, EMBO J, 12 (10):3967-3975). A recent epidemiological study in a cohort of patients with SA bacteremia shows inverse correlation between probability of sepsis and pre-existing antibodies to Hla, PSM-α3, as well as δ-toxin (Adhikari, et al., 2012, J Infect Dis, 206 (6):915-923).
Staphylococcal superantigens (SAgs) induce a massive release of cytokines and chemokines, enhanced expression and activation of adhesion molecules, increased T-cell proliferation, and ultimately T-cell apoptosis/anergy. This sequence of events can culminate in Toxic Shock Syndrome (TSS), a life threatening condition (Todd, et al., 1978, Lancet, 2 (8100):1116-1118) characterized by rash, hypotension, fever, and multisystem dysfunction (Bohach, et al., 1990, Crit Rev Microbiol, 17 (4):251-272.). A major challenge in development of multivalent S. aureus vaccines including superantigens is that there are more than 20 different SAgs and there is a wide range of variability in SAg presence in clinical isolates because most SEs are on mobile genetic elements, such as plasmids or pathogenicity islands (Staphylococcal enterotoxin K (sek), (Staphylococcal enterotoxin Q seq), lysogenic phages (Staphylococcal enterotoxin A (sea), or antibiotic resistance cassettes, like SCCmec (Staphylococcal enterotoxin H (seh) (Omoe, et al., 2002, J Clin Microbiol, 40 (3):857-862). Based on an extensive literature review encompassing over 6000 clinical isolates, the most widely represented Superantigens (Sags) appear to be toxic shock syndrome toxin 1 (TSST-1) and (Staphylococcal enterotoxin C (SEC), followed by (Staphylococcal enterotoxin A (SEA), (Staphylococcal enterotoxin D (SED), and (Staphylococcal enterotoxin B (SEB). More recent studies show the emergence of (Staphylococcal enterotoxin K (SEK) and (Staphylococcal enterotoxinQ (SEQ), primarily due to circulation of the USA300 clone. Attenuated Superantigen toxoids for (Staphylococcal enterotoxin (SEA), (Staphylococcal enterotoxin B (SEB), and TSST-1 have been designed and tested in animal models of toxic shock. These mutants are deficient in binding to MHC class II protein and therefore lack superantigenic activity (subject of U.S. Pat. Nos. 6,713,284; 6,399,332; 7,087,235; 7,750,132, 7,378,257, and 8,067,202). A simplified superantigen toxoid vaccine capable of inducing broad neutralizing antibodies is therefore highly is needed to be practical for inclusion into a multivalent S. aureus vaccine.
Phenol Soluble Modulins (PSMs) and Delta-Hemolysin(δ-Toxin):
S. aureus secretes four short (˜20 amino acids, α-type) and two longer (˜40 amino acids, β-type) cytolytic peptides, known as phenol soluble modulin (PSM) (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). In addition, SA produces δ-toxin, which is similar to the α-type PSMs. These genes are expressed in all S. aureus strains under the control of the agr system (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). Recently, a novel PSM (PSM-mec) has been also identified within the staphylococcal methicillin resistance mobile genetic element SCCmec (Chatterjee, et al., 2011, PLoS One, 6 (12):e28781; Kaito, et al., 2011, PLoS Pathog, 7 (2):e1001267) suggesting that horizontal transfer of these toxins can contribute to MRSA virulence. PSMs are lytic towards neutrophils, the first line of host defense against SA (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). Furthermore, recent studies indicate a synergistic effect on hemolytic activity of β-hemolysin (Cheung, et al., 2012, Microbes Infect, 14 (4):380-386). A key role of PSMs in pathogenesis has been shown in mouse models of bacteremia and SSTI using deletion mutants (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). Furthermore, a recent report suggests a key role for PSMs in biofilm formation (Periasamy, et al., 2012, Proc Natl Acad Sci USA, 109 (4):1281-1286). Among the PSMs, PSM-α3 plays the most prominent role in S. aureus pathogenesis (Wang, et al., 2007, Nat Med, 13 (12):1510-1514).
δ-Toxin is encoded by the hld gene located within RNAIII transcript of agr locus. RNAIII is a regulatory RNA and plays a major role in regulation of SA quorum-sensing system for the expression of various virulence genes (Novick, 2003, Mol Microbiol, 48 (6):1429-1449; Novick, et al., 1993, EMBO J, 12 (10):3967-3975). With increased expression of RNAIII, the level of extracellular δ-toxin is increased reaching almost half the amount of total exoproteins at the stationary phase (Kreger, et al., 1971, Infect Immun, 3 (3):449-465). The hld−/− mutant of the CA-MRSA strain MW2 exhibited attenuated virulence in mouse bacteremia model (Wang, et al., 2007, Nat Med, 13 (12):1510-1514). A recent study also revealed that δ-toxin increases the cell number/unit area of colony by inhibiting colony spreading, resulting in a thicker giant colony and promotes the compartmentalization of SA colonies leading to efficient colonization (Omae, et al., 2012, J Biol Chem, 287 (19):15570-15579). Thus δ-toxin also appears to modulate the physical state of SA colonies. δ-toxin also plays an important role in the escape of S. aureus from phago-endosomes of human epithelial and endothelial cells in the presence of beta-toxin (Giese, et al., 2011, Cell Microbiol, 13 (2):316-329) by acting as a costimulator of human neutrophil oxidative burst (Schmitz, et al., 1997, J Infect Dis, 176 (6): 1531-1537).
S. aureus Alpha Hemolysin (Hla):
The pore forming toxins form oligomeric beta barrel pores in the plasma membrane and play an important role for bacterial spread and survival, immune evasion and tissue destruction. SA alpha-toxin (Hla) (Bhakdi and Tranum-Jensen, 1991, Microbiol Rev, 55 (4):733-751) targets many cells such as lymphocytes, macrophages, pulmonary epithelial cells and endothelium, and erythrocytes (Bhakdi and Tranum-Jensen, 1991, Microbiol Rev, 55 (4):733-751; McElroy, et al., 1999, Infect Immun, 67 (10):5541-5544). Several lines of evidence validate Hla as a prime vaccine target for prevention of S. aureus infection or complications: i) hla is encoded by a chromosomal determinant (Brown and Pattee, 1980, Infect Immun, 30 (1):36-42), and expressed in most SA isolates (Aarestrup, et al., 1999, APMIS, 107 (4):425-430; Bhakdi and Tranum-Jensen, 1991, Microbiol Rev, 55 (4):733-751; Husmann, et al., 2009, FEBS Lett, 583 (2):337-344; Shukla, et al., 2010, J Clin Microbiol, 48 (10):3582-3592); ii) A partially attenuated Hla (H35L) and antibodies to Hla protect mice against SA pneumonia and skin infections (Kennedy, et al., 2010, J Infect Dis, 202 (7):1050-1058; Bubeck Wardenburg and Schneewind, 2008, J Exp Med, 205 (2):287-294; Ragle and Bubeck Wardenburg, 2009, J Infect Dis, 176 (6):1531-1537); iii) Antibodies to H35L protect mice from toxin and partially protect against bacterial challenge (Menzies and Kernodle, 1996; Infect Immun, 64 (5):1839-1841). While the H35 mutation largely abrogates the lytic activity of Hla, a single point mutation is not considered sufficiently safe to be developed as vaccine for human use.
WO 2012/109167A1 describes a rationally designed mutant vaccine for Hla referred to as AT62. Immunization of mice with AT62 protected the animals against S. aureus lethal sepsis and pneumonia (Adhikari, et al., 2012, PLoS One, 7 (6):e38567). Furthermore, antibodies raised against AT62 protected mice from lethal sepsis induced by S. aureus (Adhikari, et al., 2012, PLoS One, 7 (6):e38567).
Panton-Valentine Leukocidin (PVL):
PVL is a member of a family of bicomponent cytolytic toxins known as leukotoxins that is produced by several CA-MRSA lineages (Diep and Otto, 2008, Trends Microbiol, 16 (8):361-369). The bi-component hemolysins and leukotoxins, play an important role in staphylococcal immune evasion. These toxins kill key immune cells and cause tissue destruction, thereby often weakening the host during the first stage of infection and promoting bacterial dissemination and metastatic growth in distant organs. The two PVL components LukS-PV and LukF-PV are secreted separately, and form the pore-forming octameric complex upon binding of LukS-PV to its receptor and subsequent binding of LukF-PV to LukS-PV (Miles, et al., 2002, Protein Sci, 11 (4):894-902; Pedelacq, et al., 2000, Proc Natl Acad Sci USA, 109 (4):1281-1286). Targets of PVL are polymorphonuclear phagocytes (PMN), monocytes, and macrophages. Epidemiologically, PVL is associated with primary skin infections, such as furunculosis and severe necrotizing pneumonia that rapidly progresses towards acute respiratory distress syndrome. The role of PVL in skin, bone, and lung infections has been shown in animal models (Brown, et al., 2009 Clin Microbiol Infect, 15 (2):156-164; Cremieux, et al., 2009, PLoS ONE, 4 (9):e7204; Diep, et al., 2010, Proc Natl Acad Sci USA, 107 (12):5587-5592; Tseng, et al., 2009 PLoS ONE, 4 (7):e6387; Varshney, et al., 2010, J Infect Dis 1; 201(1):92-6). PVL-positive CA-MRSA affect healthy children or young adults that had neither any recent contact with health care facilities nor with any risk factors with a mortality of up to 75% (Gillet, et al., 2002, Lancet, 359 (9308):753-759; Lina, et al., 1999, Clin Infect Dis, 29 (5):1128-1132).
PCT application No. PCT/US12/67483 discloses rationally designed mutants vaccine for LukS-PV and LukF-PV. Immunization of mice with these mutants protected the animals against S. aureus lethal sepsis (Karauzum, et al., 2013, PLoS ONE, 8 (6):e65384). Furthermore, antibodies raised against LukS-PV mutant protected mice from lethal sepsis induced by S. aureus (Karauzum, et al., 2013, PLoS ONE, 8 (6):e65384).
Staphylococcus aureus Enterotoxins:
Superantigens (SAgs) constitute a large family of pyrogenic toxins composed of staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1) (Johns and Khan, 1988, J Bacteriol, 170 (9):4033-4039). In contrast to conventional antigens that undergo proteolytic processing by antigen presenting cells and are presented as MHC/peptide complex to T cells, SAgs cross link TCR with MHC Class II and activate up to 30% of T cells (Choi, et al., 1989, Proc Natl Acad Sci USA, 86 (22):8941-8; Marrack and Kappler, 1990, Science, 248 (4959):1) leading to massive release of cytokines and chemokines, enhanced expression as well as activation of cell-adhesion molecules, increased T-cell proliferation, and eventually T-cell apoptosis/anergy. This sequence of events can culminate in Toxic Shock Syndrome (TSS), a life threatening condition (Todd, et al., 1978, Lancet, 2 (8100):1116-1118) characterized by rash, hypotension, fever, and multisystem dysfunction (Bohach, et al., 1990, Crit Rev Microbiol, 17 (4):251-272). Antibodies play an important role in protection against TSS (Bonventre, et al., 1984, J Infect Dis, 150 (5):662-666; Notermans, et al., 1983, J Clin Microbiol, 18 (5):1055-1060), thus individuals that do not seroconvert towards the offending toxin due to hyporesponsive T-cells (Mahlknecht, et al., 1996, Hum Immunol, 45 (1):42-4) and/or T-cell dependent B-cell apoptosis (Hofer, et al., 1996, Proc Natl Acad Sci USA, 93 (11):5425-5430) are more likely to experience recurring bouts. Clonal deletion of CD4 T cells can further impair effective antibody response to other S. aureus antigens. Furthermore, at lower non-TSS inducing concentrations SAgs impact the virulence of S. aureus strains through induction of a local excessive inflammatory response. Attenuated mutants of SEs and TSST-1 have been developed that are deficient in binding to MHC-class II molecules. These mutants can serve as a vaccine for S. aureus infections as well as toxic shock syndrome by inducing neutralizing antibodies against superantigens.